Oligonucleotide modification, signal amplification, and nucleic acid detection by target-catalyzed product formation

ABSTRACT

A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5&#39;-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5&#39;-nuclease to cleave the oligonucleotide to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3&#39; of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte. The method has particular application to the detection of a polynucleotide analyte such as DNA. Kits for conducting methods in accordance with the present invention are also disclosed.

This is a continuation of application Ser. No. 08/691,627 filed Aug. 2,1996, now U.S. Pat. No. 5,792,614, which is a Continuation of U.S.application Ser. No. 08/363,169 filed Dec. 23, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Nucleic acid hybridization has been employed for investigating theidentity and establishing the presence of nucleic acids. Hybridizationis based on complementary base pairing. When complementary singlestranded nucleic acids are incubated together, the complementary basesequences pair to form double stranded hybrid molecules. The ability ofsingle stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA)to form a hydrogen bonded structure with a complementary nucleic acidsequence has been employed as an analytical tool in molecular biologyresearch. The availability of radioactive nucleoside triphosphates ofhigh specific activity and the ³² P labelling of DNA with T4polynucleotide kinase has made it possible to identify, isolate, andcharacterize various nucleic acid sequences of biological interest.Nucleic acid hybridization has great potential in diagnosing diseasestates associated with unique nucleic acid sequences. These uniquenucleic acid sequences may result from genetic or environmental changein DNA by insertions, deletions, point mutations, or by acquiringforeign DNA or RNA by means of infection by bacteria, molds, fungi, andviruses. Nucleic acid hybridization has, until now, been employedprimarily in academic and industrial molecular biology laboratories. Theapplication of nucleic acid hybridization as a diagnostic tool inclinical medicine is limited because of the frequently very lowconcentrations of disease related DNA or RNA present in a patient's bodyfluid and the unavailability of a sufficiently sensitive method ofnucleic acid hybridization analysis.

Current methods for detecting specific nucleic acid sequences generallyinvolve immobilization of the target nucleic acid on a solid supportsuch as nitrocellulose paper, cellulose paper, diazotized paper, or anylon membrane. After the target nucleic acid is fixed on the support,the support is contacted with a suitably labelled probe nucleic acid forabout two to forty-eight hours. After the above time period, the solidsupport is washed several times at a controlled temperature to removeunhybridized probe. The support is then dried and the hybridizedmaterial is detected by autoradiography or by spectrometric methods.

When very low concentrations must be detected, the current methods areslow and labor intensive, and nonisotopic labels that are less readilydetected than radiolabels are frequently not suitable. A method forincreasing the sensitivity to permit the use of simple, rapid,nonisotopic, homogeneous or heterogeneous methods for detecting nucleicacid sequences is therefore desirable.

Recently, a method for the enzymatic amplification of specific segmentsof DNA known as the polymerase chain reaction (PCR) method has beendescribed. This in vitro amplification procedure uses two or moredifferent oligonucleotide primers for different strands of the targetnucleic acid and is based on repeated cycles of denaturation,oligonucleotide primer annealing, and primer extension by thermophilicpolymerase, resulting in the exponential increase in copies of theregion flanked by the primers. The different PCR primers, which annealto opposite strands of the DNA, are positioned so that the polymerasecatalyzed extension product of one primer can serve as a template strandfor the other primer, leading to the accumulation of discrete fragmentswhose length is defined by the distance between the 5'-ends of theoligonucleotide primers.

Other methods for amplifying nucleic acids are single primeramplification, ligase chain reaction (LCR), nucleic acid sequence basedamplification (NASBA) and the Q-beta-replicase method. Regardless of theamplification used, the amplified product must be detected.

Depending on which of the above amplification methods are employed, themethods generally employ from seven to twelve or more reagents.Furthermore, the above methods provide for exponential amplification ofa target or a reporter oligonucleotide. Accordingly, it is necessary torigorously avoid contamination of assay solutions by the amplifiedproducts to avoid false positives. Some of the above methods requireexpensive thermal cycling instrumentation and additional reagents andsample handling steps are needed for detection of the amplified product.

Most assay methods that do not incorporate amplification of a target DNAavoid the problem of contamination, but they are not adequatelysensitive or simple. Some of the methods involve some type of sizediscrimination such as electrophoresis, which adds to the complexity ofthe methods.

One method for detecting nucleic acids is to employ nucleic acid probes.One method utilizing such probes is described in U.S. Pat. No.4,868,104, the disclosure of which is incorporated herein by reference.A nucleic acid probe may be, or may be capable of being, labeled with areporter group or may be, or may be capable of becoming, bound to asupport. Detection of signal depends upon the nature of the label orreporter group. If the label or reporter group is an enzyme, additionalmembers of the signal producing system include enzyme substrates and soforth. The product of the enzyme reaction is preferably a luminescentproduct, or a fluorescent or non-fluorescent dye, any of which can bedetected spectrophotometrically, or a product that can be detected byother spectrometric or electrometric means. If the label is afluorescent molecule, the medium can be irradiated and the fluorescencedetermined. Where the label is a radioactive group, the medium can becounted to determine the radioactive count.

It is desirable to have a sensitive, simple method for detecting nucleicacids. The method should minimize the number and complexity of steps andreagents. The need for sterilization and other steps needed to preventcontamination of assay mixtures should be avoided.

2. Description of the Related Art

Methods for detecting nucleic acid sequences are discussed by Duck, etal., in U.S. Pat. No. 5,011,769 and corresponding International PatentApplication WO 89/10415. A method of cleaving a nucleic acid molecule isdisclosed in European Patent Application 0 601 834 A1 (Dahlberg, etal.).

Holland, et al., Clinical Chemistry (1992) 38:462-463, describedetection of specific polymerase chain reaction product by utilizing the5' to 3' exonuclease activity of Thermus aguaticus DNA polymerase.Longley, et al., Nucleic Acids Research (1990) 18:7317-7322, discusscharacterization of the 5' to 3' exonuclease associated with Thermusaquaticus DNA polymerase. Lyamichev, et al., Science (1993) 260:778-783,disclose structure-specific endonucleolytic cleavage of nucleic acids byeubacterial DNA polymerases.

A process for amplifying, detecting and/or cloning nucleic acidsequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,965,188 and 5,008,182. Sequence polymerization bypolymerase chain reaction is described by Saiki, et al., (1986) Science,230: 1350-1354. Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase is described by Saiki, et al., Science(1988) 239:487.

U.S. patent applications Ser. Nos. 07/299,282, now abandoned, and07/399,795, now abandoned, filed Jan. 19, 1989, and Aug. 29, 1989,respectively, describe nucleic acid amplification using a singlepolynucleotide primer. The disclosures of these applications areincorporated herein by reference including the references listed in thesections entitled "Description of the Related Art."

Other methods of achieving the result of a nucleic acid amplificationare described by Van Brunt in Bio/Technology (1990) 8(No.4): 291-294.These methods include ligase chain reaction (LCR), nucleic acid sequencebased amplification (NASBA) and Q-beta-replicase amplification of RNA.LCR is also discussed in European Patent Applications Nos. 439,182(Backman I) and 473,155 (Backman II).

NASBA is a promoter-directed, isothermal enzymatic process that inducesin vitro continuous, homogeneous and isothermal amplification ofspecific nucleic acid.

Q-beta-replicase relies on the ability of Q-beta-replicase to amplifyits RNA substrate exponentially under isothermal conditions.

Another method for conducting an amplification of nucleic acids isreferred to as strand displacement amplification (SDA). SDA is anisothermal, in vitro DNA amplification technique based on the ability ofa restriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its restriction site and the ability of aDNA polymerase to initiate replication at the nick and displace thedownstream nontemplate strand intact. Primers containing the recognitionsites for the nicking restriction enzyme drive the exponentialamplification.

Another amplification procedure for amplifying nucleic acids is known as3SR, which is an RNA specific target method whereby RNA is amplified inan isothermal process combining promoter directed RNA polymerase,reverse transcriptase and RNase H with target RNA.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method for modifying anoligonucleotide. The method comprises incubating the oligonucleotidewith a polynucleotide and a 5'-nuclease wherein at least a portion ofthe oligonucleotide is reversibly hybridized to the polynucleotide underisothermal conditions. The oligonucleotide is cleaved to provide (i) afirst fragment that is substantially non-hybridizable to thepolynucleotide and includes no more than one nucleotide from the 5'-endof the portion and (ii) a second fragment that is 3' of the firstfragment with reference to the intact oligonucleotide and issubstantially hybridizable to the polynucleotide.

Another aspect of the present invention is a method for detecting apolynucleotide analyte. An oligonucleotide is reversibly hybridized witha polynucleotide analyte and a 5'-nuclease under isothermal conditions.The polynucleotide analyte serves as a recognition element to enable a5'-nuclease to cleave the oligonucleotide to provide (i) a firstfragment that is substantially non-hybridizable to the polynucleotideanalyte and (ii) a second fragment that lies 3' of the first fragment(in the intact oligonucleotide) and is substantially hybridizable to thepolynucleotide analyte. At least a 100-fold molar excess of the firstfragment and/or the second fragment are obtained relative to the molaramount of the polynucleotide analyte. The presence of the first fragmentand/or the second fragment is detected, the presence thereof indicatingthe presence of the polynucleotide analyte.

Another embodiment of the present invention is a method for detecting apolynucleotide analyte. A combination is provided comprising a mediumsuspected of containing the polynucleotide analyte, an excess, relativeto the suspected concentration of the polynucleotide analyte, of a firstoligonucleotide at least a portion of which is capable of reversiblyhybridizing with the polynucleotide analyte under isothermal conditions,a 5'-nuclease, and a second oligonucleotide having the characteristic ofhybridizing to a site on the polynucleotide analyte that is 3' of thesite at which the first oligonucleotide hybridizes. The polynucleotideanalyte is substantially fully hybridized to the second oligonucleotideunder such isothermal conditions. The polynucleotide is reversiblyhybridized under the isothermal conditions to the first oligonucleotide,which is cleaved as a function of the presence of the polynucleotideanalyte to provide, in at least a 100-fold molar excess of thepolynucleotide analyte, (i) a first fragment that is substantiallynon-hybridizable to the polynucleotide analyte and/or (ii) a secondfragment that lies 3' of the first fragment (in the intact firstoligonucleotide) and is substantially hybridizable to the polynucleotideanalyte. The presence of the first fragment and/or the second fragmentis detected, the presence thereof indicating the presence of thepolynucleotide analyte.

Another embodiment of the present invention is a method for detecting aDNA analyte. A combination is provided comprising a medium suspected ofcontaining the DNA analyte, a first oligonucleotide at least a portionof which is capable of reversibly hybridizing with the DNA analyte underisothermal conditions, a 5'-nuclease, and a second oligonucleotidehaving the characteristic of hybridizing to a site on the DNA analytethat is 3' of the site at which the first oligonucleotide hybridizes.The DNA analyte is substantially fully hybridized to the secondoligonucleotide under isothermal conditions. The polynucleotide analyteis reversibly hybridized to the first oligonucleotide under isothermalconditions. The first oligonucleotide is cleaved to (i) a first fragmentthat is substantially non-hybridizable to the DNA analyte and (ii) asecond fragment that lies 3' of the first fragment (in the intact firstoligonucleotide) and is substantially hybridizable to the DNA analyte.At least a 100-fold molar excess, relative to the DNA analyte, of thefirst fragment and/or the second fragment is produced. The presence ofthe first fragment and/or the second fragment is detected, the presencethereof indicating the presence of the DNA analyte.

Another embodiment of the present invention is a kit for detection of apolynucleotide. The kit comprises in packaged combination (a) a firstoligonucleotide having th characteristic that, when reversiblyhybridized under isothermal conditions to the polynucleotide, it isdegradE by a 5'-nuclease to provide (i) a first fragment that issubstantially non-hybridizable to the polynucleotide and (ii) a secondfragment that is 3' of the first fragment the first oligonucleotide) andis substantially hybridizable to the polynucleotide, (b) a secondoligonucleotide having the characteristic of hybridizing to a site onthe polynucleotide that is separated by no more than one nucleotide fromthe 3'-end of the site at which the first oligonucleotide hybridizeswherein the polynucleotide is substantially fully hybridized to thesecond oligonucleotide under the isothermal conditions, and (c) a5'-nuclease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of the present invention in which anoligonucleotide (OL) is combined with a polynucleotide analyte (PA)containing a target polynucleotide sequence (TPS) and a 5'-nuclease.After hybridizing to TPS, OL is cleaved into two fragments, DOL and LN,by the 5'-nuclease. LN contains a labeled nucleotide that can bedetected.

FIG. 2 shows another embodiment of the present invention in which theoligonucleotide (OL') has a first sequence (SOL1) that does nothybridize to the target polynucleotide sequence (TPS') of thepolynucleotide analyte (PA') and a second sequence (SOL2) thathybridizes to TPS'. The 5'-nuclease cleaves OL', which is bound to TPS',into two fragments, LNSOL1 and DSOL2.

FIG. 3 shows another embodiment of the present invention, which includesa second oligonucleotide (OL2). OL2 binds to a target polynucleotidesequence (TPS2) on the polynucleotide analyte (PA") that lies 3' of thesite of hybridization (TPS1) of a sequence (SOL2") of the firstoligonucleotide (OL"). When OL" binds to PA", it can be cleaved by a5'-nuclease to provide two fragments, DSOL2" and LNSOL1".

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention permits catalyzed cleavage of an oligonucleotidethat is modulated by a portion of a polynucleotide analyte, such as apolynucleotide, that is comprised of a target polynucleotide sequence towhich a portion of the oligonucleotide hybridizes. As such, the methodsof the present invention provide for very high sensitivity assays forpolynucleotide analytes. The methods are simple to conduct and notemperature cycling is required. Consequently, no expensive thermalcycling instrumentation is needed. Furthermore, only a few reagents areused, thus further minimizing cost and complexity of an assay. Inaddition, the absence of amplified products, which are potentialamplification targets, permits the use of less rigorous means to avoidcontamination of assay solutions by target sequences that could producefalse positives.

Before proceeding further with a description of the specific embodimentsof the present invention, a number of terms will be defined.

Polynucleotide analyte--a compound or composition to be measured that isa polymeric nucleotide, which in the intact natural state can have about20 to 500,000 or more nucleotides and in an isolated state can haveabout 30 to 50,000 or more nucleotides, usually about 100 to 20,000nucleotides, more frequently 500 to 10,000 nucleotides. Isolation ofanalytes from the natural state, particularly those having a largenumber of nucleotides, frequently results in fragmentation. Thepolynucleotide analytes include nucleic acids from any source inpurified or unpurified form including DNA (dsDNA and ssDNA) and RNA,including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplastDNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes,plasmids, the genomes of biological material such as microorganisms,e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,humans, and fragments thereof, and the like. Preferred polynucleotideanalytes are double stranded DNA (dsDNA) and single stranded DNA(ssDNA). The polynucleotide analyte can be only a minor fraction of acomplex mixture such as a biological sample. The analyte can be obtainedfrom various biological material by procedures well known in the art.Some examples of such biological material by way of illustration and notlimitation are disclosed in Table I below.

                  TABLE I                                                         ______________________________________                                        Microorganisms of interest include:                                           ______________________________________                                        Corynebacteria                                                                Corynebacterium diphtheria                                                    Pneumococci                                                                   Diplococcus pneumoniae                                                        Streptococci                                                                  Streptococcus pyrogenes                                                       Streptococcus salivarus                                                       Staphylococci                                                                 Staphylococcus aureus                                                         Staphylococcus albus                                                          Neisseria                                                                     Neisseria meningitidis                                                        Neisseria gonorrhea                                                           Enterobacteriaciae                                                            Escherichia coli                                                              Aerobacter aerogenes                                                                            The colliform                                               Klebsiella pneumoniae                                                                           bacteria                                                    Salmonella typhosa                                                            Salmonella choleraesuis                                                                         The Salmonellae                                             Salmonella typhimurium                                                        Shigella dysenteria                                                           Shigella schmitzii                                                            Shigella arabinotarda                                                                           The Shigellae                                               Shigella flexneri                                                             Shigella boydii                                                               Shigella sonnei                                                               Other enteric bacilli                                                         Proteus vulgaris                                                              Proteus mirabilis Proteus species                                             Proteus morgani                                                               Pseudomonas aeruginosa                                                        Alcaligenes faecalis                                                          Vibrio cholerae                                                               Hemophilus-Bordetella group                                                                     Rhizopus oryzae                                             Hemophilus influenza, H. ducryi                                                                 Rhizopus arrhizua Phycomycetes                              Hemophilus hemophilus                                                                           Rhizopus nigricans                                          Hemophilus aegypticus                                                                           Sporotrichum schenkii                                       Hemophilus parainfluenza                                                                        Flonsecaea pedrosoi                                         Bordetella pertussis                                                                            Fonsecacea compact                                          Pasteurellae      Fonsecacea dermatidis                                       Pasteurella pestis                                                                              Cladosporium carrionii                                      Pasteurella tulareusis                                                                          Phialophora verrucosa                                       Brucellae         Aspergillus nidulans                                        Brucella melitensis                                                                             Madurella mycetomi                                          Brucella abortus  Madurella grisea                                            Brucella suis     Allescheria boydii                                          Aerobic Spore-forming Bacilli                                                                   Phialophora jeanselmei                                      Bacillus anthracis                                                                              Microsporum gypseum                                         Bacillus subtilis Trichophyton mentagrophytes                                 Bacillus megaterium                                                                             Keratinomyces ajelloi                                       Bacillus cereus   Microsporum canis                                           Anaerobic Spore-forming Bacilli                                                                 Trichophyton rubrum                                         Clostridium botulinum                                                                           Microsporum adouini                                         Clostridium tetani                                                                              Viruses                                                     Clostridium perfringens                                                                         Adenoviruses                                                Clostridium novyi Herpes Viruses                                              Clostridium septicum                                                                            Herpes simplex                                              Clostridium histolyticum                                                                        Varicella (Chicken pox)                                     Clostridium tertium                                                                             Herpes Zoster (Shingles)                                    Clostridium bifermentans                                                                        Virus B                                                     Clostridium sporogenes                                                                          Cytomegalovirus                                             Mycobacteria      Pox Viruses                                                 Mycobacterium tuberculosis                                                                      Variola (smallpox)                                          hominis                                                                       Mycobacterium bovis                                                                             Vaccinia                                                    Mycobacterium avium                                                                             Poxvirus bovis                                              Mycobacterium leprae                                                                            Paravaccinia                                                Mycobacterium paratuberculosis                                                                  Molluscum contagiosum                                       Actinomycetes                                                                 (fungus-like bacteria)                                                                          Picornaviruses                                              Actinomyces Isaeli                                                                              Poliovirus                                                  Actinomyces bovis Coxsackievirus                                              Actinomyces naeslundii                                                                          Echoviruses                                                 Nocardia asteroides                                                                             Rhinoviruses                                                Nocardia brasiliensis                                                                           Myxoviruses                                                 The Spirochetes   Influenza (A, B, and C)                                     Treponema pallidum                                                                       Spirillum  Parainfluenza (1-4)                                                minus                                                              Treponema pertenue                                                                       Streptobacillus                                                                          Mumps Virus                                                        monoiliformis                                                                            Newcastle Disease Virus                                 Treponema carateum                                                                              Measles Virus                                               Borrelia recurrentis                                                                            Rinderpest Virus                                            Leptospira icterohemorrhagiae                                                                   Canine Distemper Virus                                      Leptospira canicola                                                                             Respiratory Syncytial Virus                                 Trypanasomes      Rubella Virus                                               Mycoplasmas       Arboviruses                                                 Mycoplasma pneumoniae                                                         Other pathogens   Eastern Equine Encephalitis Virus                           Listeria monocytogenes                                                                          Western Equine Encephalitis Virus                           Erysipelothrix rhusiopathiae                                                                    Sindbis Virus                                               Streptobacillus moniliformis                                                                    Chikugunya Virus                                            Donvania granulomatis                                                                           Semliki Forest Virus                                        Bartonella bacilliformis                                                                        Mayora Virus                                                Rickettsiae (bacteria-like                                                                      St. Louis Encephalitis Virus                                parasites)                                                                    Rickettsia prowazekii                                                                           California Encephalitis Virus                               Rickettsia mooseri                                                                              Colorado Tick Fever Virus                                   Rickettsia rickettsii                                                                           Yellow Fever Virus                                          Rickettsia conori Dengue Virus                                                Rickettsia australis                                                                            Reoviruses                                                  Rickettsia sibiricus                                                                            Reovirus Types 1-3                                                            Retroviruses                                                Rickettsia akari  Human Immunodeficiency Viruses                              (HIV)                                                                         Rickettsia tsutsugamushi                                                                        Human T-cell Lymphotrophic                                                    Virus I & II (HTLV)                                         Rickettsia burnetti                                                                             Hepatitis                                                   Rickettsia quintana                                                                             Hepatitis A Virus                                           Chlamydia (unclassifiable parasites                                                             Hepatitis B Virus                                           bacterial/viral)  Hepatitis nonA-nonB Virus                                   Chlamydia agents                                                              (naming uncertain)                                                                              Tumor Viruses                                               Fungi             Rauscher Leukemia Virus                                     Cryptococcus neoformans                                                                         Gross Virus                                                 Blastomyces dermatidis                                                                          Maloney Leukemia Virus                                      Hisoplasma capsulatum                                                         Coccidioides immitis                                                                            Human Papilloma Virus                                       Paracoccidioides brasiliensis                                                 Candida albicans                                                              Aspergillus fumigatus                                                         Mucor corymbifer (Absidia corymbifera)                                        ______________________________________                                    

The polynucleotide analyte, where appropriate, may be treated to cleavethe analyte to obtain a polynucleotide that contains a targetpolynucleotide sequence, for example, by shearing or by treatment with arestriction endonuclease or other site specific chemical cleavagemethod. However, it is an advantage of the present invention that thepolynucleotide analyte can be used in its isolated state without furthercleavage.

For purposes of this invention, the polynucleotide analyte, or a cleavedpolynucleotide obtained from the polynucleotide analyte, will usually beat least partially denatured or single stranded or treated to render itdenatured or single stranded. Such treatments are well-known in the artand include, for instance, heat or alkali treatment. For example, doublestranded DNA can be heated at 90-100° C. for a period of about 1 to 10minutes to produce denatured material.

3'- or 5'-End of an oligonucleotide--as used herein this phrase refersto a portion of an oligonucleotide comprising the 3'- or 5'- terminus,respectively, of the oligonucleotide.

3'- or 5'-Terminus of an oligonucleotide--as used herein this termrefers to the terminal nucleotide at the 3'- or 5'- end, respectively,of an oligonucleotide.

Target polynucleotide sequence--a sequence of nucleotides to beidentified, which may be the polynucleotide analyte but is usuallyexisting within a polynucleotide comprising the polynucleotide analyte.The identity of the target polynucleotide sequence is known to an extentsufficient to allow preparation of an oligonucleotide having a portionor sequence that hybridizes with the target polynucleotide sequence. Ingeneral, when one oligonucleotide is used, the oligonucleotidehybridizes with the 5'-end of the target polynucleotide sequence. When asecond oligonucleotide is used, it hybridizes to a site on the targetpolynucleotide sequence that is 3' of the site to which the firstoligonucleotide hybridizes. (It should be noted that the relationshipcan be considered with respect to the double stranded molecule formedwhen the first and second oligonucleotides are hybridized to thepolynucleotide. In such context the second oligonucleotide is5-primeward of the first oligonucleotide with respect to the "strand"comprising the first and second oligonucleotides.) The relationshipsdescribed above are more clearly seen with reference to FIG. 3. Thetarget polynucleotide sequence usually contains from about 10 to 1,000nucleotides, preferably 15 to 100 nucleotides, more preferably, 20 to 70nucleotides. The target polynucleotide sequence is part of apolynucleotide that may be the entire polynucleotide analyte. Theminimum number of nucleotides in the target polynucleotide sequence isselected to assure that the presence of target polynucleotide sequencein a sample is a specific indicator of the presence of polynucleotideanalyte in a sample. Very roughly, the sequence length is usuallygreater than about 1.6 log L nucleotides where L is the number of basepairs in the genome of the biologic source of the sample. The number ofnucleotides in the target sequence is usually the sum of the lengths ofthose portions of the oligonucleotides that hybridize with the targetsequence plus the number of nucleotides lying between the portions ofthe target sequence that hybridize with the oligonucleotides.

Oligonucleotide--a polynucleotide, usually a synthetic polynucleotide,usually single stranded that is constructed such that at least a portionthereof hybridizes with the target polynucleotide sequence of thepolynucleotide. The oligonucleotides of this invention are usually 10 to150 nucleotides, preferably, deoxyoligonucleotides of 15 to 100nucleotides, more preferably, 20 to 60 nucleotides, in length.

The first oligonucleotide, or "the" oligonucleotide when a secondoligonucleotide is not employed, has a 5'-end about 0 to 100nucleotides, preferably, 1 to 20 nucleotides in length that does nothybridize with the target polynucleotide sequence and usually has a 10to 40 nucleotide sequence that hybridizes with the target polynucleotidesequence. In general, the degree of amplification is reduced somewhat asthe length of the portion of the oligonucleotide that does not hybridizewith the target polynucleotide sequence increases. The firstoligonucleotide also may have a sequence at its 3'-end that does nothybridize with the target polynucleotide sequence.

The second oligonucleotide preferably hybridizes at its 3'-end with thetarget polynucleotide sequence at a site on the target polynucleotidesequence 3' of the site of binding of the first oligonucleotide. Thelength of the portion of the second oligonucleotide that hybridizes withthe target polynucleotide sequence is usually longer than the length ofthe portion of the first oligonucleotide that hybridizes with the targetpolynucleotide sequence and is usually 20 to 100 nucleotides. Themelting temperature of the second oligonucleotide hybridized to thetarget polynucleotide sequence is preferably at least as high, morepreferably, at least 5° C. higher than the melting temperature of thefirst oligonucleotide hybridized to the target polynucleotide sequence.

The oligonucleotides can be oligonucleotide mimics such apolynucleopeptides, phosphorothioates or phosphonates except that thefirst oligonucleotide usually has at least one phosphodiester bond tothe nucleoside at the 5'-end of the sequence that hybridizes with thetarget polynucleotide sequence. When oligonucleotide mimics are usedthat provide very strong binding, such as polynucleopeptides, the lengthof the portion of the second oligonucleotide that hybridizes with thetarget polynucleotide sequence may be reduced to less than 20 and,preferably, greater than 10.

Various techniques can be employed for preparing an oligonucleotide orother polynucleotide utilized in the present invention. They can beobtained by biological synthesis or by chemical synthesis. For shortoligonucleotides (up to about 100 nucleotides) chemical synthesis willfrequently be more economical as compared to biological synthesis. Inaddition to economy, chemical synthesis provides a convenient way ofincorporating low molecular weight compounds and/or modified basesduring the synthesis step. Furthermore, chemical synthesis is veryflexible in the choice of length and region of the target polynucleotidesequence. The oligonucleotides can be synthesized by standard methodssuch as those used in commercial automated nucleic acid synthesizers.Chemical synthesis of DNA on a suitably modified glass or resin resultsin DNA covalently attached to the surface. This may offer advantages inwashing and sample handling. For longer sequences standard replicationmethods employed in molecular biology can be used such as the use of M13for single stranded DNA as described by J. Messing (1983) MethodsEnzymol, 101, 20-78.

In addition to standard cloning techniques, in vitro enzymatic methodsmay be used such as polymerase catalyzed reactions. For preparation ofRNA, T7 RNA polymerase and a suitable DNA template can be used. For DNA,polymerase chain reaction (PCR) and single primer amplification areconvenient.

Other chemical methods of polynucleotide or oligonucleotide synthesisinclude phosphotriester and phosphodiester methods (Narang, et al.,Meth. Enzymol (1979) 68: 90) and synthesis on a support (Beaucage, etal., Tetrahedron (1981) Letters 22: 1859-1862) as well asphosphoramidate techniques, Caruthers, M. H., et al., "Methods inEnzymology," Vol. 154, pp. 287-314 (1988), and others described in"Synthesis and Applications of DNA and RNA," S. A. Narang, editor,Academic Press, New York, 1987, and the references contained therein.

Fragment--in general, in the present method the oligonucleotide (or thefirst oligonucleotide when a second oligonucleotide is employed) iscleaved only when at least a portion thereof is reversibly hybridizedwith a target polynucleotide sequence and, thus, the targetpolynucleotide sequence acts as a recognition element for cleavage ofthe oligonucleotide, thereby yielding two portions. One fragment issubstantially non-hybridizable to the target polynucleotide sequence.The other fragment is substantially hybridizable to the targetpolynucleotide sequence and 3' of the other fragment with respect to theoligonucleotide in its uncleaved form.

5'-Nuclease--a sequence-independent deoxyribonuclease enzyme thatcatalyzes the cleavage of an oligonucleotide into fragments only when atleast a portion of the oligonucleotide is hybridized to the targetpolynucleotide sequence. The enzyme selectively cleaves theoligonucleotide near the 5'-terminus of the bound portion, within 5nucleotides thereof, preferably within 1 to 2 nucleotides thereof anddoes not cleave the unhybridized oligonucleotide or the targetpolynucleotide sequence. Such enzymes include both 5'-exonucleases and5'-endonucleases but exclude ribonucleases such as RNAse H andrestriction enzymes. 5'-nucleases useful in the present invention mustbe stable under the isothermal conditions used in the present method andare usually thermally stable nucleotide polymerases having5'-exonuclease activity such as Taq DNA polymerase (e.g. AmpliTaq(TM)from Perkin-Elmer Corporation, Norwalk, N.J.), Thermalase Tbr(TM) DNApolymerase (from Amresco, Solon, Ohio), Ultra Therm(TM) DNA polymerase(from Bio/Can Scientific, Ontario, Canada), Replitherm(TM) DNApolymerase (from Epicentre, Madison, Wis.), Tfl(TM) DNA polymerase (fromEpicentre), Panozyme(TM) DNA polymerase (from Panorama Research,Mountain View, Calif.), Tth(TM) DNA polymerase (from Epicentre),rBst(TM) DNA polymerase (from Epicentre), Heat Tuff(TM) DNA polymerase(from Clontech, Palo Alto, Calif.), and the like, derived from anysource such as cells, bacteria, such as E. coli, plants, animals, virus,thermophilic bacteria, and so forth wherein the polymerase may bemodified chemically or through genetic engineering to provide forthermal stability and/or increased activity.

Isothermal conditions--a uniform or constant temperature at which themodification of the oligonucleotide in accordance with the presentinvention is carried out. The temperature is chosen so that the duplexformed by hybridizing the oligonucleotide to a polynucleotide with atarget polynucleotide sequence is in equilibrium with the free orunhybridized oligonucleotide and free or unhybridized targetpolynucleotide sequence, a condition that is otherwise referred toherein as "reversibly hybridizing" the oligonucleotide with apolynucleotide. Normally, at least 1%, preferably 20 to 80%, usuallyless than 95% of the polynucleotide is hybridized to the oligonucleotideunder the isotermal conditions. Accordingly, under isothermal conditionsthere are molecules of polynucleotide that are hybridized with theoligonucleotide, or portions thereof, and are in dynamic equilibriumwith molecules that are not hybridized with the oligonucleotide. Somefluctuation of the temperature may occur and still achieve the benefitsof the present invention. The fluctuation generally is not necessary forcarrying out the methods of the present invention and usually offer nosubstantial improvement. Accordingly, the term "isothermal conditions"includes the use of a fluctuating temperature, particularly random oruncontrolled fluctuations in temperature, but specifically excludes thetype of fluctuation in temperature referred to as thermal cycling, whichis employed in some known amplification procedures, e.g., polymerasechain reaction.

Polynucleotide primer(s) or oligonucleotide primer(s)--anoligonucleotide that is usually employed in a chain extension on apolynucleotide template.

Nucleoside triphosphates--nucleosides having a 5'-triphosphatesubstituent. The nucleosides are pentose sugar derivatives ofnitrogenous bases of either purine or pyrimidine derivation, covalentlybonded to the 1'-carbon of the pentose sugar, which is usually adeoxyribose or a ribose. The purine bases include adenine(A),guanine(G), inosine, and derivatives and analogs thereof. The pyrimidinebases include cytosine (C), thymine (T), uracil (U), and derivatives andanalogs thereof. Nucleoside triphosphates include deoxyribonucleosidetriphosphates such as dATP, dCTP, dGTP and dTTP and ribonucleosidetriphosphates such as rATP, rCTP, rGTP and rUTP. The term "nucleosidetriphosphates" also includes derivatives and analogs thereof.

Nucleotide--a base-sugar-phosphate combination that is the monomericunit of nucleic acid polymers, i.e., DNA and RNA.

Nucleoside--is a base-sugar combination or a nucleotide lacking aphosphate moiety.

Nucleotide polymerase--a catalyst, usually an enzyme, for forming anextension of an oligonucleotide along a polynucleotide template wherethe extension is complementary thereto. The nucleotide polymerase is atemplate dependent polynucleotide polymerase and utilizes nucleosidetriphosphates as building blocks for extending the 3'-end of aoligonucleotide to provide a sequence complementary with the singlestranded portion of the polynucleotide to which the oligonucleotide ishybridized to form a duplex.

Hybridization (hybridizing) and binding--in the context of nucleotidesequences these terms are used interchangeably herein. The ability oftwo nucleotide sequences to hybridize with each other is based on thedegree of complementarity of the two nucleotide sequences, which in turnis based on the fraction of matched complementary nucleotide pairs. Themore nucleotides in a given sequence that are complementary to anothersequence, the more stringent the conditions can be for hybridization andthe more specific will be the binding of the two sequences. Increasedstringency is achieved by elevating the temperature, increasing theratio of cosolvents, lowering the salt concentration, and the like.

Homologous or substantially identical--In general, two polynucleotidesequences that are identical or can each hybridize to the samepolynucleotide sequence are homologous. The two sequences are homologousor substantially identical where the sequences each have at least 90%,preferably 100%, of the same or analogous base sequence where thymine(T) and uracil (U) are considered the same. Thus, the ribonucleotides A,U, C and G are taken as analogous to the deoxynucleotides dA, dT, dC,and dG, respectively. Homologous sequences can both be DNA or one can beDNA and the other RNA.

Complementary--Two sequences are complementary when the sequence of onecan bind to the sequence of the other in an anti-parallel sense whereinthe 3'-end of each sequence binds to the 5'-end of the other sequenceand each A, T(U), G, and C of one sequence is then aligned with a T(U),A, C, and G, respectively, of the other sequence.

Copy--means a sequence that is a direct identical or homologous copy ofa single stranded polynucleotide sequence as differentiated from asequence that is complementary to the sequence of such single strandedpolynucleotide.

Member of a specific binding pair ("sbp member")--one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to, and is thereby defined as complementary with, aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These may be members of an immunological pairsuch as antigen-antibody, or may be operator-repressor,nuclease-nucleotide, biotin-avidin, hormones-hormone receptors, nucleicacid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like.

Ligand--any compound for which a receptor naturally exists or can beprepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors,protection enzymes, protein A, complement component Clq, DNA bindingproteins or ligands and the like.

Small organic molecule--a compound of molecular weight less than 1500,preferably 100 to 1000, more preferably 300 to 600 such as biotin,fluorescein, rhodamine and other dyes, tetracycline and other proteinbinding molecules, and haptens, etc. The small organic molecule canprovide a means for attachment of a nucleotide sequence to a label or toa support or may itself be a label.

Support or surface--a porous or non-porous water insoluble material. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Binding of sbp members to a support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface canhave any one of a number of shapes, such as strip, rod, particle,including bead, and the like.

Label or reporter group or reporter molecule--a member of a signalproducing system. Usually the label or reporter group or reportermolecule is conjugated to or becomes bound to, or fragmented from, anoligonucleotide or to a nucleoside triphosphate and is capable of beingdetected directly or, through a specific binding reaction, and canproduce a detectible signal. In general, any label that is detectablecan be used. The label can be isotopic or nonisotopic, usuallynon-isotopic, and can be a catalyst, such as an enzyme or a catalyticpolynucleotide, promoter, dye, fluorescent molecule, chemiluminescer,coenzyme, enzyme substrate, radioactive group, a small organic molecule,amplifiable polynucleotide sequence, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectible group,and the like. Labels include an oligonucleotide or specificpolynucleotide sequence that can provide a template for amplification orligation or act as a ligand such as for a repressor protein. The labelis a member of a signal producing system and can generate a detectablesignal either alone or together with other members of the signalproducing system. The label can be bound directly to a nucleotidesequence or can become bound thereto by being bound to an sbp membercomplementary to an sbp member that is bound to a nucleotide sequence.

Signal Producing System--The signal producing system may have one ormore components, at least one component being the label or reportergroup or reporter molecule. The signal producing system generates asignal that relates to the presence or amount of target polynucleotidesequence or a polynucleotide analyte in a sample. The signal producingsystem includes all of the reagents required to produce a measurablesignal. When the label is not conjugated to a nucleotide sequence, thelabel is normally bound to an sbp member complementary to an sbp memberthat is bound to, or part of, a nucleotide sequence. Other components ofthe signal producing system may be included in a developer solution andcan include substrates, enhancers, activators, chemiluminescentcompounds, cofactors, inhibitors, scavengers, metal ions, specificbinding substances required for binding of signal generating substances,and the like. Other components of the signal producing system may becoenzymes, substances that react with enzymic products, other enzymesand catalysts, and the like. The signal producing system provides asignal detectable by external means, by use of electromagneticradiation, desirably by visual examination. The signal-producing systemis described more fully in U.S. patent application Ser. No. 07/555,323,filed Jul. 19, 1990, now U.S. Pat. No. 5,595,891, the relevantdisclosure of which is incorporated herein by reference.

Amplification of nucleic acids or polynucleotides--any method thatresults in the formation of one or more copies of a nucleic acid or apolynucleotide molecule, usually a nucleic acid or polynucleotideanalyte, or complements thereof, present in a medium.

Exponential amplification of nucleic acids or polynucleotides--anymethod that results in the formation of one or more copies of a nucleicacid or polynucleotide molecule, usually a nucleic acid orpolynucleotide analyte, present in a medium.

Methods for the enzymatic amplification of specific double strandedsequences of DNA include those described above such as the polymerasechain reaction (PCR), amplification of a single stranded polynucleotideusing a single polynucleotide primer, ligase chain reaction (LCR),nucleic acid sequence based amplification (NASBA), Q-beta-replicasemethod, strand displacement amplification (SDA), and 3SR.

Conditions for carrying out an amplification, thus, vary depending uponwhich method is selected. Some of the methods such as PCR utilizetemperature cycling to achieve denaturation of duplexes, oligonucleotideprimer annealing, and primer extension by thermophilic templatedependent polynucleotide polymerase. Other methods such as NASBA,Q-beta-replicase method, SDA and 3SR are isothermal. As can be seen,there are a variety of known amplification methods and a variety ofconditions under which these methods are conducted to achieveexponential amplification.

Linear amplification of nucleic acids or polynucleotides--any methodthat results in the formation of one or more copies of only thecomplement of a nucleic acid or polynucleotide molecule, usually anucleic acid or polynucleotide analyte, present in a medium. Thus, onedifference between linear amplification and exponential amplification isthat the latter produces copies of the polynucleotide whereas the formerproduces only the complementary strand of the polynucleotide. In linearamplification the number of complements formed is, in principle,directly proportional to the time of the reaction as opposed toexponential amplification wherein the number of copies is, in principle,an exponential function of the time or the number of temperature cycles.

Ancillary Materials--various ancillary materials will frequently beemployed in the methods and assays carried out in accordance with thepresent invention. For example, buffers will normally be present in theassay medium, as well as stabilizers for the assay medium and the assaycomponents. Frequently, in addition to these additives, proteins may beincluded, such as albumins, organic solvents such as formamide,quaternary ammonium salts, polycations such as dextran sulfate,surfactants, particularly non-ionic surfactants, binding enhancers,e.g., polyalkylene glycols, or the like.

As mentioned above, the present invention has a primary application tomethods for detecting a polynucleotide analyte. In one aspect of theinvention an oligonucleotide is reversibly hybridized with apolynucleotide analyte in the presence of a 5'-nuclease under isothermalconditions. In this way the polynucleotide analyte serves as a"recognition element" to enable the 5'-nuclease to specifically cleavethe oligonucleotide to provide first and second fragments when theoligonucleotide is reversibly hybridized to the polynucleotide analyte.The first fragment comprises the 5'-end of the oligonucleotide (withreference to the intact or original oligonucleotide) and issubstantially non-hybridizable to the polynucleotide analyte and canserve as a label. The first fragment generally includes at least aportion of that part the 5'-end of the original oligonucleotide that wasnot hybridized to the polynucleotide analyte when the portion of theoligonucleotide that is hybridizable with the polynucleotide analyte isreversibly hybridized thereto. Additionally, the first fragment mayinclude nucleotides (usually, no more than 5, preferably, no more than2, more preferably, no more than 1 of such nucleotides) that are cleavedby the 5'-nuclease from the 5'-end of that portion (or sequence) of theoriginal oligonucleotide that was hybridized to the polynucleotideanalyte. Therefore, it is in the above context that the first fragmentis "substantially non-hybridizable" with the polynucleotide analyte. Thesecond fragment comprises the sequence of nucleotides at the 3'-end ofthe oligonucleotide that were reversibly hybridized to thepolynucleotide analyte minus those nucleotides cleaved by the5'-nuclease when the original oligonucleotide is reversibly hybridizedto the polynucleotide analyte. Accordingly, the second fragment is"substantially hybridizable" to the polynucleotide analyte havingresulted from that portion of the oligonucleotide that reversiblyhybridizes with the polynucleotide analyte.

As mentioned above, the 3'-end of the oligonucleotide may include one ormore nucleotides that do not hybridize with the polynucleotide analyteand may comprise a label. At least a 100-fold molar excess of the firstfragment and/or the second fragment are obtained relative to the molaramount of the polynucleotide analyte. The sequence of at least one ofthe fragments is substantially preserved during the reaction. Thepresence of the first fragment and/or the second fragment is detected,the presence thereof indicating the presence of the polynucleotideanalyte.

The 5'-nuclease is generally present in an amount sufficient to causethe cleavage of the oligonucleotide, when it is reversibly hybridized tothe polynucleotide analyte, to proceed at least half as rapidly as themaximum rate achievable with excess enzyme, preferably, at least 75% ofthe maximum rate. The concentration of the 5'-nuclease is usuallydetermined empirically. Preferably, a concentration is used that issufficient such that further increase in the concentration does notdecrease the time for the amplification by over 5-fold, preferably2-fold. The primary limiting factor generally is the cost of thereagent. In this respect, then, the polynucleotide analyte, or at leastthe target polynucleotide sequence, and the enzyme are generally presentin a catalytic amount.

The oligonucleotide that is cleaved by the enzyme is usually in largeexcess, preferably, 10⁻⁹ M to 10⁻⁵ M, and is used in an amount thatmaximizes the overall rate of its cleavage in accordance with thepresent invention wherein the rate is at least 10%, preferably, 50%,more preferably, 90%, of the maximum rate of reaction possible.Concentrations of the oligonucleotide lower than 50% may be employed tofacilitate detection of the fragment(s) produced in accordance with thepresent invention. The amount of oligonucleotide is at least as great asthe number of molecules of product desired. Usually, the concentrationof the oligonucleotide is 0.1 nanomolar to 1 millimolar, preferably, 1nanomolar to 10 micromolar. It should be noted that increasing theconcentration of the oligonucleotide causes the reaction rate toapproach a limiting value that depends on the oligonucleotide sequence,the temperature, the concentration of the target polynucleotide sequenceand the enzyme concentration. For many detection methods very highconcentrations of the oligonucleotide may make detection more difficult.

The amount of the target polynucleotide sequence that is to be copiedcan be as low as one or two molecules in a sample but generally may varyfrom about 10² to 10¹⁰, more usually from about 10³ to 10⁸ molecules ina sample preferably at least 10⁻²¹ M in the sample and may be 10⁻¹⁰ to10⁻¹⁹ M, more usually 10⁻¹⁴ to 10⁻¹⁹ M.

In carrying out the methods in accordance with the present invention, anaqueous medium is employed. Other polar solvents may also be employed ascosolvents, usually oxygenated organic solvents of from 1-6, moreusually from 1-4, carbon atoms, including alcohols, ethers and the like.Usually these cosolvents, if used, are present in less than about 70weight percent, more usually in less than about 30 weight percent.

The pH for the medium is usually in the range of about 4.5 to 9.5, moreusually in the range of about 5.5-8.5, and preferably in the range ofabout 6-8. The pH and temperature are chosen so as to achieve thereversible hybridization or equilibrium state under which cleavage of anoligonucleotide occurs in accordance with the present invention. In someinstances, a compromise is made in the reaction parameters in order tooptimize the speed, efficiency, and specificity of these steps of thepresent method. Various buffers may be used to achieve the desired pHand maintain the pH during the determination. Illustrative buffersinclude borate, phosphate, carbonate, Tris, barbital and the like. Theparticular buffer employed is not critical to this invention but inindividual methods one buffer may be preferred over another.

As mentioned above the reaction in accordance with the present inventionis carried out under isothermal conditions. The reaction is generallycarried out at a temperature that is near the melting temperature of theoligonucleotide:polynucleotide analyte complex. Accordingly, thetemperature employed depends on a number of factors. Usually, forcleavage of the oligonucleotide in accordance with the presentinvention, the temperature is about 35° C. to 90° C. depending on thelength and sequence of the oligonucleotide. It will usually be desiredto use relatively high temperature of 60° C. to 85° C. to provide for ahigh rate of reaction. The amount of the fragments formed depends on theincubation time and temperature. In general, a moderate temperature isnormally employed for carrying out the methods. The exact temperatureutilized also varies depending on the salt concentration, pH, solventsused, and the length of and composition of the target polynucleotidesequence as well as the oligonucleotide as mentioned above.

One embodiment of the invention is depicted in FIG. 1. OligonucleotideOL is combined with polynucleotide analyte PA having targetpolynucleotide sequence TPS and with a 5'-nuclease, which can be, forexample, a Taq polymerase. In this embodiment OL is labeled (*) withinwhat is designated the first fragment, produced upon cleavage of theoligonucleotide in accordance with the present invention. OL in thisembodiment usually is at least 10 nucleotides in length, preferably,about 10 to 50 nucleotides in length, more preferably, 15 to 30 or morenucleotides in length. In general, the length of OL should be sufficientso that a portion hybridizes with TPS, the length of such portionapproximating the length of TPS. In this embodiment the length of OL ischosen so that the cleavage of no more than 5, preferably, no more than1 to 3, more preferably, 1 to 2 nucleotides, therefrom results in twofragments. The first fragment, designated LN, is no more than 5nucleotides in length, preferably, 1 to 3 nucleotides in length, morepreferably, 1 to 2 nucleotides in length and the second fragment,designated DOL, is no more than 5, preferably, no more than 1 to 3, morepreferably, no more than 1 to 2, nucleotides shorter than the length ofOL.

As shown in FIG. 1, OL hybridizes with TPS to give duplex I. Thehybridization is carried out under isothermal conditions so that OL isreversibly hybridized with TPS. OL in duplex I is cleaved to give DOLand LN, wherein LN includes a labeled nucleotide (*) In the embodimentdepicted in FIG. 1, DOL is the complement of TPS except for thenucleotides missing at the 5'-end. Since during the course of theisothermal reaction the 5'-end of PA may be cleaved at or near the5'-end of TPS, DOL may also have 0 to 5 nucleotides at its 3'-end thatoverhang and cannot hybridize with the residual portion of TPS. Theisothermal conditions are chosen such that equilibrium exists betweenduplex I and its single stranded components, namely, PA and OL. Uponcleavage of OL within duplex I, an equilibrium is also establishedbetween duplex I and its single stranded components, PA and DOL. SinceOL is normally present in large excess relative to the amount of DOLformed in the reaction, there are usually many more duplexes containingOL than DOL. The reaction described above for duplex I continuouslyproduces additional molecules of DOL.

The reaction is allowed to continue until a sufficient number ofmolecules of DOL and LN are formed to permit detection of the labeled LN(LN*) and, thus, the polynucleotide analyte. In this way theenzyme-catalyzed cleavage of nucleotides from the 5'-end of OL ismodulated by and, therefore, related to the presence of thepolynucleotide analyte. Depending on the amount of PA present, asufficient number of molecules for detection can be obtained where thetime of reaction is from about 1 minute to 24 hours. Preferably, thereaction can be carried out in less than 5 hours. As a matter ofconvenience it is usually desirable to minimize the time period as longas the requisite of number of molecules of detectable fragment isachieved. In general, the time period for a given degree of cleavage canbe minimized by optimizing the temperature of the reaction and usingconcentrations of the 5'-nuclease and the oligonucleotide that providereaction rates near the maximum achievable with excess of thesereagents. Detection of the polynucleotide analyte is accomplishedindirectly by detecting the label in fragment LN*. Alternatively, DOLmay be detected, for example, by using the label as a means ofseparating LN* and OL from the reaction mixture and then detecting theresidual DOL.

Detection of the labeled fragment is facilitated in a number of ways.For example, a specific pair member such as biotin or a directlydetectable label such a fluorescein can be used. The low molecularweight LN* can be separated by electrophoresis, gel exclusionchromatography, thin layer chromatography ultrafiltration and the likeand detected by any convenient means such as a competitive binding assayor direct detection of the label. Alternatively, the oligonucleotide canbe labeled within the second (DOL) fragment with a specific bindingmember such as a ligand, a small organic molecule, a polynucleotidesequence or a protein, or with a directly detectable label such as adirectly detectable small organic molecules, e.g., fluorescein, asensitizer, a coenzyme and the like. Detection will then depend ondifferentiating the oligonucleotide with labels on both ends from singlylabeled fragments where one labeled end has been cleaved. In this caseit is desirable to label one end of OL with a specific binding memberthat facilitates removal of OL and the fragment retaining the label byusing a complementary sbp member bound to a support. The residuallabeled fragments bearing the other label are then detected by using amethod appropriate for detecting that label.

One method for detecting nucleic acids is to employ nucleic acid probes.Other assay formats and detection formats are disclosed in U.S. patentapplications Ser. Nos. 07/299,282, now abandoned, and 07/399,795, nowabandoned, filed Jan. 19, 1989, and Aug. 29, 1989, respectively, U.S.patent application Ser. No. 07/555,323 filed Jul. 19, 1990, now U.S.Pat. No. 5,595,891, U.S. patent application Ser. No. 07/555,968, nowU.S. Pat. No. 5,439,193, and U.S. patent application Ser. No. 07/776,538filed Oct. 11, 1991, now abandoned, which have been incorporated hereinby reference.

Examples of particular labels or reporter molecules and their detectioncan be found in U.S. patent application Ser. No. 07/555,323 filed Jul.19, 1990, now U.S. Pat. No. 5,595,891, the relevant disclosure of whichis incorporated herein by reference.

Detection of the signal will depend upon the nature of the signalproducing system utilized. If the label or reporter group is an enzyme,additional members of the signal producing system include enzymesubstrates and so forth. The product of the enzyme reaction ispreferably a luminescent product, or a fluorescent or non-fluorescentdye, any of which can be detected spectrophotometrically, or a productthat can be detected by other spectrometric or electrometric means. Ifthe label is a fluorescent molecule, the medium can be irradiated andthe fluorescence determined. Where the label is a radioactive group, themedium can be counted to determine the radioactive count.

Another embodiment of the present invention is depicted in FIG. 2.Oligonucleotide OL' has a first portion or sequence SOL1 that is nothybridized to TPS' and a second portion or sequence SOL2 that ishybridized to TPS'. OL' is combined with polynucleotide analyte PA'having target polynucleotide sequence TPS' and with a 5'-endonuclease(5'-endo), which can be, for example, Taq DNA polymerase and the like.OL' and 5'-endo are generally present in concentrations as describedabove. In the embodiment of FIG. 2, OL' is labeled (*) within thesequence SOL1 wherein SOL1 may intrinsically comprise the label or maybe extrinsically labeled with a specific binding member or directlydetectable labeled. The length of SOL2 is as described in the embodimentof FIG. 1. In general, the length of SOL2 should be sufficient tohybridize with TPS', usually approximating the length of TPS'. SOL1 maybe any length as long as it does not substantially interfere with thecleavage of OL' and will preferably be relatively short to avoid suchinterference. Usually, SOL1 is about 1 to 100 nucleotides in length,preferably, 8 to 20 nucleotides in length.

In this embodiment the cleavage of SOL1 from SOL2 results in twofragments. Cleavage in SOL2 occurs within 5 nucleotides of the bondjoining SOL1 and SOL2 in OL'. The exact location of cleavage is notcritical so long as the enzyme cleaves OL' only when it is bound toTPS'. The two fragments are designated LNSOL1 and DSOL2. LNSOL1 iscomprised of the 5'-end of OL' and DSOL2 is comprised of the 3'-end ofOL'. The sequence of at least one of LNSOL1 and DSOL2 remainssubstantially intact during the cleavage reaction. As shown in FIG. 2,SOL2 of OL' hybridizes with TPS' to give duplex I'. The hybridization iscarried out under isothermal conditions so that OL' is reversiblyhybridized with TPS'. OL' in duplex I' is cleaved to give DSOL2 andLNSOL1, the latter of which comprises a label. In the embodimentdepicted in FIG. 2, DSOL2 is the complement of TPS' except for anynucleotides missing at the 5'-end thereof as a result of the cleavage ofthe cleavage reaction and any nucleotides appended to the 3'-end of OL'(not shown in FIG. 2) that do not hybridize with TPS'.

The isothermal conditions are chosen such that equilibrium existsbetween duplex I' and its single stranded components, i.e., PA' and OL'.Upon cleavage of OL' within duplex I' and equilibrium is alsoestablished between duplex I' and its single stranded components, PA'and DSOL2. Since OL' is normally present in large excess relative to theamount of DSOL2 formed in the reaction, there are usually many moreduplexes containing OL' than DSOL2. The reaction described above forduplex I' continuously produces molecules of DSOL2 and LNSOL1. Thereaction is allowed to continue until a sufficient number of moleculesof DSOL2 and LNSOL1 are formed to permit detection of one or both ofthese fragments. In this way the enzyme-catalyzed cleavage of LNSOL1from the 5'-end of the portion of OL' hybridized to PA' is modulated by,and therefore related to, the presence of the polynucleotide analyte.The reaction parameters and the detection of DSOL2 and/or LNSOL1 aregenerally as described above for the embodiment of FIG. 1.

Various ways of controlling the cleavage of the oligonucleotide can beemployed. For example, the point of cleavage can be controlled byintroducing a small organic group, such as biotin, into the nucleotideat the 5'-terminus of OL' or the nucleotide in SOL2 that is at thejunction of SOL2 and SOL1.

An embodiment using a second oligonucleotide is depicted in FIG. 3. Thesecond oligonucleotide (OL2) hybridizes to a site TPS2 on PA" that lies3' of the site of hybridization (TPS1) of the sequence SOL2" of thefirst oligonucleotide, namely, OL". In the embodiment shown OL2 fullyhybridizes with TPS2. This is by way of example and not limitation. Thesecond oligonucleotide can include nucleotides at its 5' end that arenot hybridizable with the target polynucleotide sequence, but its 3'-endis preferably hybridizable. Preferably, OL2 binds to a site (TPS2) thatis contiguous with the site to which SOL2" hybridizes (TPS1). However,it is within the purview of the present invention that the secondoligonucleotide hybridize with PA" within 1 to 5 nucleotides,preferably, 1 nucleotide, of the site to which SOL2" hybridizes. Thesecond oligonucleotide, OL2, is usually at least as long as, andpreferably longer than, SOL2", preferably, at least 2 nucleotides longerthan SOL2". In general, the second oligonucleotide is about 20-100nucleotides in length, preferably, 30-80 nucleotides in length dependingon the length of SOL2". Normally, the second oligonucleotide is chosensuch that it dissociates from duplex I" at a higher temperature thanthat at which OL" dissociates, usually at least 3° C., preferably, atleast 5° C. or more higher.

The presence of OL2 in duplex I" can effect the site of cleavage of OL".In particular, when OL2 binds to PA" that is not contiguous with theSOL2" site of hybridization, the cleavage site may be shifted one ormore nucleotides.

The concentration of the second oligonucleotide employed in thisembodiment is usually at least 1 picomolar, but is preferably above 0.1nanomolar to facilitate rapid binding to PA", more preferably, at least1 nanomolar to 1 micromolar. In accordance with the embodiment of FIG.3, OL" in duplex I" is cleaved by 5'-endo to give DSOL2" and LNSOL1".The reaction is permitted to continue until the desired number ofmolecules of labeled fragment are formed. The reaction parameters anddetection of DSOL2" and/or LNSOL1" are similar to those described abovefor the embodiment of FIG. 1.

In general and specifically in any of the embodiments of FIGS. 1 to 3above, the 3'-end of the first oligonucleotide, for example, OL, OL' andOL", may have one or more nucleotides that do not hybridize with thetarget polynucleotide sequence and can serve as a label but need not doso.

It is also within the purview of the present invention to employ asingle nucleoside triphosphate in any of the above embodiments,depending on the particular 5'-endonuclease chosen for the abovecleavage. The decision to use a nucleoside triphosphate and the choiceof the nucleoside triphosphate are made empirically based on its abilityto accelerate the reaction in accordance with the present invention. Thenucleoside triphosphate is preferably one that cannot be incorporatedinto the first oligonucleotide as a consequence of the binding of theoligonucleotide to the target polynucleotide sequence. In thisparticular embodiment the added nucleoside triphosphate is present in aconcentration of 1 micromolar to millimolar, preferably, 10 micromolarto 1 millimolar, more preferably, 100 micromolar to 1 millimolar. It isalso withih the purview of the present invention to utilize the addednucleoside triphosphate to chain extend the 3'-terminus of the secondoligonucleotide to render it contiguous with the site on the targetpolynucleotide sequence at which the first oligonucleotide hybridizes.In this approach the second oligonucleotide serves as a polynucleotideprimer for chain extension. In addition, the nucleoside triphosphate isappropriately selected to accomplish such chain extension and the5'-nuclease is selected to also have template-dependent nucleotidepolymerase activity. In any event such an approach is primarilyapplicable to the situation where the site of binding of this secondoligonucleotide, TPS2, is separated from the site of binding of thefirst oligonucleotide, TPS1, by a sequence of one or more identicalbases that are complementary to the added nucleotide triphosphate.

In the embodiment of FIG. 3 the mixture containing PA", OL", the secondoligonucleotide OL2 and the nucleoside triphosphate is incubated at anappropriate isothermal temperature at which OL" and PA" are inequilibrium with duplex I" wherein most of the molecules of PA" andduplex I" are hybridized to OL2. During the time when a molecule of OL"is bound to PA", the 5'-endo causes the cleavage by hydrolysis of OL" inaccordance with the present invention. When the remaining portion ofcleaved oligonucleotide (DSOL2") dissociates from PA", an intactmolecule of OL" becomes hybridized, whereupon the process is repeated.

In one experiment in accordance with the above embodiment, incubationfor 3 hours at 72° C. resulted in the production of over 10¹² moleculesof DSOL2" and LNSOL1", which was over 10⁴ increase over the number ofmolecules of PA" that was present initially in the reaction mixture. OL"was labeled with a ³² P-phosphate at the 5'-terminus. The cleavedproduct LNSOL1" was detected by applying the mixture to anelectrophoresis gel and detecting a band that migrated more rapidly thanthe band associated with OL". The appearance of this band was shown tobe associated with the presence and amount of PA" where a minimum of 10⁸molecules of PA" was detected.

Alternative approaches for detection of LNSOL1" and/or DSOL2" may alsobe employed in the above embodiment. For example, in one approach biotinis attached to any part of SOL2" that is cleaved from OL" by the5'-endonuclease. The fragment DSOL2" and OL" containing the biotin areseparated from LNSOL1", for example, by precipitation with streptavidinand filtration. The unprecipitated labeled fragment LNSOL1" is thendetected by any standard binding assay, either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products.

Homogeneous immunoassays are exemplified by enzyme multipliedimmunoassay techniques ("EMIT") disclosed in Rubenstein, et al., U.S.Pat. No. 3,817,837, column 3, line 6 to column 6, line 64;immunofluorescence methods such as those disclosed in Ullman, et al.,U.S. Pat. No. 3,996,345, column 17, line 59 to column 23, line 25;enzyme channeling techniques such as those disclosed in Maggio, et al.,U.S. Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; andother enzyme immunoassays such as the enzyme linked immunosorbant assay("ELISA") are discussed in Maggio, E. T. supra. Exemplary ofheterogeneous assays are the radioimmunoassay, disclosed in Yalow, etal., J. Clin. Invest. 39:1157 (1960). The above disclosures are allincorporated herein by reference. For a more detailed discussion of theabove immunoassay techniques, see "Enzyme-Immunoassay," by Edward T.Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. See also, for example,U.S. Pat. Nos. 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987;3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,996,345; and 4,098,876,which listing is not intended to be exhaustive.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive assay formats are disclosed in Davalian, et al., U.S.Pat. No. 5,089,390, column 14, line 25 to column 15, line 9,incorporated herein by reference. A typical non-competitive assay is asandwich assay disclosed in David, et al., U.S. Pat. No. 4,486,530,column 8, line 6 to column 15, line 63, incorporated herein byreference.

Another binding assay approach involves the luminescent immunoassaydescribed in U.S. Ser. No. 07/704,569, filed May 22, 1991 entitled"Assay Method Utilizing Induced Luminescence", which disclosure isincorporated herein by reference.

As a matter of convenience, predetermined amounts of reagents employedin the present invention can be provided in a kit in packagedcombination. A kit can comprise in packaged combination (a) a firstoligonucleotide having the characteristic that, when reversiblyhybridized to a portion of a polynucleotide to be detected, it isdegraded under isothermal conditions by a 5'-nuclease to provide (i) afirst fragment that is substantially non-hybridizable to thepolynucleotide and (ii) a second fragment that is 3' of the firstfragment and is substantially hybridizable to the polynucleotide, (b) asecond oligonucleotide having the characteristic of at least a portionthereof hybridizing to a site on the polynucleotide that is 3' of thesite at which the first oligonucleotide hybridizes wherein thepolynucleotide is substantially fully hybridized to such portion of thesecond oligonucleotide under isothermal conditions, and (c) the above5'-nuclease. The kit can further comprise a single nucleosidetriphosphate.

The above kits can further include members of a signal producing systemand also various buffered media, some of which may contain one or moreof the above reagents. The above kits can also include a writtendescription of one or more of the methods in accordance with the presentinvention for detecting a polynucleotide analyte.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents which substantiallyoptimize the reactions that need to occur during the present method andto further substantially optimize the sensitivity of any assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay inaccordance with the present invention. Each reagent can be packaged inseparate containers or some reagents can be combined in one containerwhere cross-reactivity and shelf life permit.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. Temperatures are in degrees centigrade (° C.) and parts andpercentages are by weight, unless otherwise indicated.

Example 1

A single stranded target DNA (2×10⁸ molecules) (M13mp19 from Gibco, BRL,Bethesda, Md.) (the "target DNA") was combined with a 5'³² P-labeledoligonucleotide probe, Probe 1, (10 uM) (5'CGT-GGG-AAC-AAA-CGG-CGG-AT3'(SEQ ID NO:1) synthesized on a Pharmacia Gene Assembler (PharmaciaBiotech, Piscataway, N.J.), an unlabeled oligonucleotide, Probe 2, (1uM) (5'TTC-ATC-AAC-ATT-AAA-TGT-GAG-CGA-GTA-ACA-ACC-CGT-CGG-ATT-CTC3'(SEQ ID NO:2) synthesized on a Pharmacia Gene Assembler (PharmaciaBiotech), and 7.5 units of AmpliTaq DNA polymerase (from Perkin-ElmerCorporation, Norwalk, N.J.) in 50 uL of buffer (10 mM Tris-HCl, pH 8.5,50 mM KCl, 7.5 mM MgCl₂, 100 uM dATP) Probe 1 was a 20-baseoligonucleotide that was fully complementary to the target DNA and had alabel on the 5'-nucleotide. Probe 2, the unlabeled probe, was designedto anneal to the target DNA 3' to, and contiguous with, the site atwhich the labeled probe annealed to the target DNA. The dATP was shownto enhance the rate of cleavage by the polymerase. However, good resultswere obtained in the absence of dATP.

The reaction mixture was incubated at 72° C. and accumulation ofproduct, a mononucleotide, namely, 5'³² P-C--OH, was determined byvisualization using autoradiography following polyacrylamide gelelectrophoresis. The fold of amplification was determined by liquidscintillation spectrometry of excised reaction products. A 10⁵ foldamplification was observed.

The above reaction protocol was repeated using, in place of Probe 1, alabeled probe, Probe 3, (5'TCG-TGG-GAA-CAA-ACG-GCG-GAT3' (SEQ ID NO:3)prepared using a Pharmacia Gene Assembler) that had 21 nucleotides withone base at the 5'-end that was not complementary, and did not hybridizewith, the target DNA. The product of this reaction was a dinucleotide,namely, 5'³² P-TC--OH (SEQ ID NO:4), that represented a 10⁵ -foldamplification.

The above reaction protocol was repeated with different temperatures anddifferent concentrations of reagents. All of the reactions, includingthose mentioned above, were carried out for a period of 3 hours. Thefollowing table summarizes the reagents and reaction parameters and theresults obtained during the optimization procedure.

    __________________________________________________________________________       Probe                                                                            Target                                                                            Taq                      Fold                                       Probe                                                                            (μM)                                                                          number                                                                            (units)                                                                           Temp ° C.                                                                   Conditions      amplification                              __________________________________________________________________________    1  1  .sup. 10.sup.10                                                                   2.5 72   buffer as described; 1.5 mM MgCl.sub.2                                                        8.8 × 10.sup.2                          1  10.sup.9                                                                          |                                                                        |                                                                         |      1.8 × 10.sup.3                          1  10.sup.8                                                                          ↓                                                                          |                                                                         |      N.D.*                                         1  10.sup.9                                                                          7.5 |                                                                         ↓        2.0 × 10.sup.3                          1  10.sup.9                                                                          |                                                                        |                                                                         add dATP(100 μM)                                                                           1.4 × 10.sup.3                          1  10.sup.8                                                                          |                                                                        |                                                                         |      1.0 × 10.sup.4                          10 10.sup.9                                                                          |                                                                        |                                                                         |      1.4 × 10.sup.4                          10 10.sup.8                                                                          |                                                                        |                                                                         ↓        3.6 × 10.sup.4                          1  10.sup.9                                                                          |                                                                        |                                                                         increase MgCl.sup.2 (7.5 mM)                                                                  9.7 × 10.sup.3                          1  10.sup.8                                                                          |                                                                        |                                                                         |      1.2 × 10.sup.4                          1  10.sup.9                                                                          |                                                                        |                                                                         |      9.3 × 10.sup.3                          1  10.sup.8                                                                          |                                                                        |                                                                         |      2.8 × 10.sup.4                          1  10.sup.7                                                                          |                                                                        ↓                                                                           |      N.D.*                                         10 10.sup.9                                                                          |                                                                        74   |      3.7 × 10.sup.4                          10 10.sup.8                                                                          |                                                                        |                                                                         |      1.1 × 10.sup.5                          10 10.sup.7                                                                          |                                                                        ↓                                                                           |      N.D.*                                      3  1  10.sup.9                                                                          |                                                                        72   |      9.9 × 10.sup.3                          1  10.sup.8                                                                          |                                                                        |                                                                         |      2.6 × 10.sup.4                          1  10.sup.7                                                                          |                                                                        ↓                                                                           |      N.D.*                                         10 10.sup.9                                                                          |                                                                        74   |      4.6 × 10.sup.4                          10 10.sup.8                                                                          |                                                                        |                                                                         |      1.0 × 10.sup.5                          10 10.sup.7                                                                          ↓                                                                          ↓                                                                           ↓        N.D.*                                      __________________________________________________________________________     *N.D. = not detected                                                     

Example 2

The reaction protocol described in Example 1 was repeated using thefollowing probes in place of Probe 1 or Probe 3:

Probe 4: 5'TTA-TTT-CGT-GGG-AAC-AAA-CGG-CGG-AT3' (SEQ ID NO:5) (fromOligos Etc., Inc., Wilsonville, Oreg.). Probe 4 had 26 nucleotides withsix nucleotides at its 5'-end that were not complementary, norhybridizable with, the target DNA. Probe 4 was present in aconcentration of 1 micromolar. The product of this reaction was anintact seven nucleotide fragment, namely, 5'³² P-TTATTTC-OH (SEQ IDNO:6), that represented a 1.5×10⁴ -fold amplification.

Probe 5: 5'GAT-TAG-GAT-TAG-GAT-TAG-TCG-TGG-GAA-CAA-ACG-GCG-GAT3' (SEQ IDNO:7) was prepared using a Pharmacia Gene assembler and had 39nucleotides with 19 nucleotides at its 5'-end that were notcomplementary and did not hybridize with the target DNA. The product ofthis reaction was an intact 20 nucleotide fragment, namely, 5'³²P-GAT-TAG-GAT-TAG-GAT-TAG-TC-OH (SEQ ID NO:8), that represented a1.5×10⁴ -fold amplification.

In repeating the above reactions in the absence of Probe 2, product wasobserved but the intensity of the spot on the polyacrylamide gel wassignificantly less than in the presence of Probe 2. Similar results werealso observed where a 1 nucleotide space existed between the 3'-end ofProbe 2 and the second probe when both probes were hybridized to thetarget DNA.

Example 3

The reaction protocol described in Example 1 was repeated using 2×10⁹target molecules and Probe 5 (see Example 2) at a concentration of 1micromolar in place of Probe 1. The reactions were conducted for threehours at a temperature of 72° C. using one of six different DNApolymerases, namely, AmpliTaq DNA polymerase, Replitherm(TM) DNApolymerase (Epicentre), Tfl(TM) DNA polymerase (Epicentre), UltraTherm(TM) DNA polymerase (Bio/Can Scientific), Thermalase Tbr (TM) DNApolymerase (Amresco) and Panozyme(TM) DNA polymerase. The product of thereaction was a 20-nucleotide fragment (see Example 2). The following isa summary of the results obtained.

    ______________________________________                                        Enzyme       Fragment (picomoles)                                             ______________________________________                                        AmpliTaq     32                                                               Replitherm   18                                                               Tfl           5                                                               Ultra Therm  27                                                               Tbr          16                                                               Panozyme     25                                                               ______________________________________                                    

The above experiments demonstrate that detectable cleavage products weregenerated in a target-specific manner at a single temperature usingenzymes having 5'-nuclease activity and a labeled oligonucleotide. Theaccumulation of product was enhanced by the presence of a secondoligonucleotide that was longer than the first labeled oligonucleotideand that was annealed to the target polynucleotide sequence 3' of thesite of hybridization of the first labeled oligonucleotide. Thereactions were carried out at temperatures very close to the meltingtemperature (Tm) of the labeled oligonucleotide with the targetpolynucleotide sequence.

The above discussion includes certain theories as to mechanisms involvedin the present invention. These cheories should not be construed tolimit the present invention in any way, since it has been demonstratedthat the present invention achieves the results described.

The above description and examples fully disclose the inventionincluding preferred embodiments thereof. Modifications of the methodsdescribed that are obvious to those of ordinary skill in the art such asmolecular biology and related sciences are intended to be within thescope of the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 20               GGAT                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #45                GTGA GCGAGTAACA ACCCGTCGGA TTCTC                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #21                CGGA T                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #              26  AACG GCGGAT                                                - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #    39            AGTC GTGGGAACAA ACGGCGGAT                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 # 20               AGTC                                                       __________________________________________________________________________

What is claimed is:
 1. A method for modifying an oligonucleotide, saidmethod comprising:(a) combining said oligonucleotide with apolynucleotide and a 5'-nuclease, said oligonucleotide having a 3'portion capable of reversibly hybridizing to said polynucleotide and a5'-portion which does not hybridize to the polynucleotide, (b)incubating said oligonucleotide, said polynucleotide and said nucleaseunder isothermal conditions, whereby a duplex formed by hybridization ofthe 3' portion of the oligonucleotide to the polynucleotide is inequilibrium with unhybridized oligonucleotide and unhybridizedpolynucleotide, said isothermal conditions being at or near the meltingtemperature of said complex, and (c) while maintaining said isothermalconditions, cleaving said oligonucleotide with said nuclease when said3'-portion is hybridized to said polynucleotide to provide:(i) a firstfragment including said 5'-portion and no more than one nucleotide fromthe 5'-end of said 3'-portion, and (ii) a second fragment that is 3' ofsaid first fragment with reference to the intact oligonucleotide,thereby modifying said oligonucleotide, wherein said first fragment andsaid second fragment are continuously produced under said isothermalconditions.
 2. The method of claim 1, wherein the amounts of fragmentsthat are formed are at least 100-fold larger than the amount of saidpolynucleotide.
 3. The method of claim 1, further comprising incubatinga second oligonucleotide under said isothermal conditions with saidoligonucleotide, said polynucleotide, and said 5'-nuclease, wherein saidsecond oligonucleotide substantially non-reversibly hybridizes undersaid isothermal conditions to a site on said polynucleotide that is inthe 3' direction from the site at which said oligonucleotide hybridizes.4. The method according to claim 3, wherein the melting temperature ofthe second oligonucleotide when hybridized to the polynucleotide is atleast 3° C. higher than the melting temperature of the firstoligonucleotide when hybridized to the polynucleotide.
 5. A method foramplifying a signal associated with the presence of a polynucleotideanalyte, said method comprising:(a) providing in combination apolynucleotide analyte, a 5'-nuclease and a molar excess, relative tothe concentration of said polynucleotide analyte, of an oligonucleotidehaving a 3' portion capable of reversibly hybridizing to saidpolynucleotide and a 5'-portion which does not hybridize to saidpolynucleotide, (b) under isothermal conditions, establishing anequilibrium between said oligonucleotide, said polynucleotide analyte,and a duplex formed by the hybridization of the 3' portion of saidoligonucleotide with said polynucleotide analyte, said isothermalconditions being at or near the melting temperature of said duplex, (c)while maintaining said isothermal conditions, cleaving saidoligonucleotide with said 5'-nuclease when said oligonucleotide ishybridized to said polynucleotide to provide,(i) a first fragmentincluding said 5'-portion and no more than one nucleotide from the5'-end of said 3'-portion, and (ii) a second fragment including at leastone of said 3' portion and said 3' portion lacking onenucleotide,wherein at least one of said first fragment and said secondfragment generates a signal, and (d) while maintaining said isothermalconditions, maintaining said equilibrium to amplify the amount of atleast one of said first fragment and said second fragment and therebyamplifying said signal, wherein said first fragment and said secondfragment are continuously produced under said isothermal conditions. 6.The method of claim 5 further comprising maintaining said equilibriumuntil at least a 100-fold molar excess of said first fragment and/orsaid second fragment are obtained relative to the molar amount of saidpolynucleotide analyte.
 7. The method of claim 5 wherein saidpolynucleotide analyte is from a source selected from the groupconsisting of Corynebacteria, Pneumococci, Streptococci, Staphylococci,Neisseria, Enterobacteriaciae, Enteric bacilli, Hemophilus-Bordetella,Pasteurellae, Brucellae, Aerobic Spore-forming Bacilli, AnaerobicSpore-forming Bacilli, Mycobacteria, Actinomycetes, Spirochetes,Trypanasomes, Mycoplasmas, Listeria monocytogenes, Erysipelothrixrhusiopathiae, Streptobaccillus moniliformis, Donvania granulomatis,Bartonella bacilliformis, Rickettsiae, Adenoviruses, Herpes Viruses, PoxViruses, Picornaviruses, Myxoviruses, Arboviruses, Reoviruses,Retroviruses, Fungi, Hepatitis Viruses, and Tumor Viruses.
 8. The methodof claim 5, further comprising hybridizing a second oligonucleotide tosaid polynucleotide analyte under said isothermal conditions, whereinsaid second oligonucleotide hybridizes to a site on said polynucleotideanalyte that is in the 3' direction of the site at which saidoligonucleotide hybridizes, and wherein the melting temperature of thesecond oligonucleotide when hybridized to the polynucleotide is at least3° C. higher than the melting temperature of the first oligonucleotidewhen hybridized to the polynucleotide.
 9. The method of claim 5, whereinsaid oligonucleotide hybridization sites are contiguous.
 10. The methodof claim 5, wherein at least one of said first fragment and said secondfragment has a label.
 11. The method of claim 10, wherein said label isselected from the group consisting of a member of a specific bindingpair, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzymesubstrates, radioactive groups, and suspendible particles.
 12. A methodfor detecting a polynucleotide analyte, said method comprising:(a)providing in combination a medium suspected of containing saidpolynucleotide analyte, a molar excess, relative to the suspectedconcentration of said polynucleotide analyte, of a first oligonucleotidehaving a 3' portion capable of reversibly hybridizing to saidpolynucleotide and a 5'-portion which does not hybridize to saidpolynucleotide, a 5'-nuclease, and a second oligonucleotide thathybridizes to a site on said polynucleotide analyte in the 3'-directionof the site at which said first oligonucleotide hybridizes, (b) underisothermal conditions, establishing an equilibrium between a complexformed by the hybridization of the 3' portion of said firstoligonucleotide and said polynucleotide analyte, said polynucleotideanalyte and said first oligonucleotide, said isothermal conditions beingat or near the melting temperature of said complex, and wherein saidsecond oligonucleotide is substantially fully hybridized to saidpolynucleotide analyte under said isothermal conditions, (c) whilemaintaining said isothermal conditions, cleaving said firstoligonucleotide when hybridized to said polynucleotide analyte with said5'-nuclease to provide,(i) a first fragment that is substantiallynon-hybridizable to said polynucleotide analyte, and (ii) a secondfragment that is 3' of said first fragment in said first oligonucleotideand which substantially hybridizes to said polynucleotide analyte; and(d) while maintaining said isothermal conditions, detecting the presenceof said first fragment, said second fragment, or said first fragment andsaid second fragment, the presence thereof indicating the presence ofsaid polynucleotide analyte wherein said first fragment and said secondfragment are continuously produced under said isothermal conditions. 13.The method according to claim 12, wherein the melting temperature of thesecond oligonucleotide when hybridized to the polynucleotide is at least3° C. higher than the melting temperature of the first oligonucleotidewhen hybridized to the polynucleotide.
 14. The method of claim 12,wherein said first fragment and/or said second fragment has a label. 15.The method of claim 14, wherein said label is selected from the groupconsisting of a member of a specific binding pair, dyes, fluorescentmolecules, chemiluminescers, coenzymes, enzyme substrates, radioactivegroups, and suspendible particles.
 16. The method of claim 12 whereinsaid polynucleotide analyte is DNA.
 17. The method of claim 12, whereinsaid first fragment includes no more than 1 nucleotide from the 5'-endof that portion of said first oligonucleotide that is capable ofhybridizing to said polynucleotide analyte.
 18. The method of claim 12,wherein said second oligonucleotide hybridizes to said polynucleotide ata site contiguous with the site on said polynucleotide at which saidfirst oligonucleotide hybridizes.
 19. The method of claim 12, whereinsaid first oligonucleotide has a substituent that facilitates separationof said first fragment or said second fragment from said medium.
 20. Themethod of claim 12 wherein said polynucleotide analyte is from a sourceselected from the group consisting of Corynebacteria, Pneumococci,Streptococci, Staphylococci, Neisseria, Enterobacteriaciae, Entericbacilli, Hemophilus-Bordetella, Pasteurellae, Brucellae, AerobicSpore-forming Bacilli, Anaerobic Spore-forming Bacilli, Mycobacteria,Actinomycetes, Spirochetes, Trypanasomes, Mycoplasmas, Listeriamonocytogenes, Erysipelothrix rhusiopathiae, Streptobaccillusmoniliformis, Donvania granulomatis, Bartonella bacilliformis,Rickettsiae, Adenoviruses, Herpes Viruses, Pox Viruses, Picornaviruses,Myxoviruses, Arboviruses, Reoviruses, Retroviruses, Fungi, HepatitisViruses, and Tumor Viruses.
 21. A method for detecting a polynucleotideanalyte, said method comprising:(a) providing in combination a mediumsuspected of containing said polynucleotide analyte, a firstoligonucleotide at least a portion of which reversibly hybridizes withsaid polynucleotide analyte under isothermal conditions to form acomplex, said isothermal conditions being at or near the meltingtemperature of said complex, a 5'-nuclease, and a second oligonucleotidethat hybridizes to a site on said polynucleotide analyte that is in 3'of, and contiguous with, the site at which said first oligonucleotidehybridizes, wherein the melting temperature of the secondoligonucleotide when hybridized to the polynucleotide is at least 3° C.higher than the melting temperature of the first oligonucleotide whenhybridized to the polynucleotide, (b) reversibly hybridizing under saidisothermal conditions said polynucleotide analyte and said firstoligonucleotide, wherein said first oligonucleotide, when hybridized tosaid polynucleotide analyte, is cleaved by said 5'-nuclease as afunction of the presence of said polynucleotide analyte to provide, inat least a 100-fold molar excess of said polynucleotide analyte,(i) afirst fragment that is substantially non-hybridizable to saidpolynucleotide analyte, and (ii) a second fragment that is 3' of saidfirst fragment in said first oligonucleotide and which substantiallyhybridizes to said polynucleotide analyte; and (c) detecting thepresence of said first fragment, said second fragment, or said firstfragment and said second fragment, the presence thereof indicating thepresence of said polynucleotide analyte, wherein said polynucleotideanalyte is from a source selected from the group consisting ofCorynebacteria, Pneumococci, Streptococci, Staphylococci, Neisseria,Enterobacteriaciae, Enteric bacilli, Hemophilus-Bordetella,Pasteurellae, Brucellae, Aerobic Spore-forming Bacilli, AnaerobicSpore-forming Bacilli, Mycobacteria, Actinomycetes, Spirochetes,Trypanasomes, Mycoplasmas, Listeria monocytogenes, Erysipelothrixrhusiopathiae, Streptobaccillus moniliformis, Donvania granulomatis,Bartonella bacilliformis, Rickettsiae, Adenoviruses, Herpes Viruses, PoxViruses, Picornaviruses, Myxoviruses, Arboviruses, Reoviruses,Retroviruses, Fungi, Hepatitis Viruses, and Tumor Viruses.
 22. Themethod of claim 21, wherein at least one of said first fragment and saidsecond fragment has a label.
 23. The method of claim 22, wherein saidlabel is selected from the group consisting of a member of a specificbinding pair, dyes, fluorescent molecules, chemiluminescers, coenzymes,enzyme substrates, radioactive groups, and suspendible particles. 24.The method of claim 22, wherein said polynucleotide analyte is DNA.